Mammalian hematopoiesis is based on the activity of hematopoietic stem cells (HSC) that undergo continuous self-renewal throughout the adult life. The long-term maintenance of bone marrow HSC appears to be regulated by highly specific transcriptional mechanisms that remain largely unknown. The progeny of self-renewing HSC initiate ordered differentiation programs that give rise to all blood lineages. In particular, the development of B and T lymphocytes involves a massive proliferative expansion of immature precursors. Both HSC self-renewal and lymphocyte expansion appear exquisitely sensitive to elevated levels of the cell] cycle inhibitor p16lnk4a, which is actively repressed by poorly defined molecular pathways. Our studies of hematopoiesis focused on Zfx, an evolutionarily conserved zinc finger-containing transcription factor. In preliminary experiments, inactivation of murine Zfx in the hematopoietic system leads to a specific loss of adult bone marrow HSC population. In addition, the expansion of immature B and T lymphocytes was severely impaired. Preliminary expression analysis suggests that these phenotypes might be caused by the overexpression of p16lnk4a in the absence of Zfx. Therefore, Zfx might represent an essential and specific positive regulator of HSC self-renewal and of immature lymphocyte expansion upstream of p16lnk4a. To test this hypothesis, three Specific Aims are proposed. First, the role of Zfx in HSC self-renewal will be characterized at the functional level. Second, early development and growth of Zfx-deficient lymphocytes will be investigated. Third, the molecular basis of Zfx activity will be established through the identification and functional analysis of its target genes in hematopoietic cells. These studies might identify novel molecular mechanisms that facilitate the maintenance of HSC and of immature lymphocytes, possibly through the repression of p16lnk4a.